Genus-specific PCR Primers Targeting Intracellular Parasite Euduboscquella (Dinoflagellata: Syndinea)
We designed a genus-specific primer pair targeting the intracellular parasite Euduboscquella. To increase target specificity and inhibit untargeted PCR, two nucleotides were added at the 3’ end of the reverse primer, one being a complementary nucleotide to the Euduboscquella-specific SNP (single-nucleotide polymorphism) and the other a deliberately mismatched nucleotide. Target specificity of the primer set was verified experimentally using PCR of two Euduboscquella species (positive controls) and 15 related species (negative controls composed of ciliates, diatoms and dinoflagellates), and analytical comparison with SILVA SSU rRNA gene database (release 119) in silico. In addition, we applied the Euduboscquella-specific primer set to four environmental samples previously determined by cytological staining to be either positive or negative for Euduboscquella. As expected, only positive controls and environmental samples known to contain Euduboscquella were successfully amplified by the primer set. An inferred SSU rRNA gene phylogeny placed environmental samples containing aloricate ciliates infected by Euduboscquella in a cluster discrete from Euduboscquella groups a–d previously reported from loricate, tintinnid ciliates.
Keywordsmarine parasite syndinean dinoflagellate SNP target-specific PCR primer ciliates
Unable to display preview. Download preview PDF.
- Akselman R, Santinelli N (1989) Observaciones sobre dinoflagelados parasitos en el litoral Atlantico sudoccidental. Physis B Aires Sec A 47:43–44Google Scholar
- Cachon J (1964) Contribution à l’étude des péridiniens parasites. Cytologie, cycles évolutifs. Ann Sci Nat Zool 6:1–158Google Scholar
- Campbell AS (1927) Studies on the marine ciliate Favella (Jörgensen), with special regard to the neuromotor appartus and its rôle in the formation of the lorica. Univ Calif Publ Zool 29:429–452Google Scholar
- Chatton É (1952) Classe des dinoflagellés ou péridiniens. In: Grassé P-P (ed) Traité de zoologie. Masson & Cie, Paris, pp 309–390Google Scholar
- Coats DW, Bachvaroff TR (2013) Parasites of tintinnids. In: Dolan JR, Montagnes DJS, Agatha S, Coats DW, Stoecker DK (eds) The biology and ecology of tintinnid ciliates: models for marine plankton. John Wiley, Chichester, pp 145–170Google Scholar
- Hall T (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acid S 41:95–98Google Scholar
- Jung J-H, Choi JM, Coats DW, Kim Y-O (2016) Euduboscquella costata n. sp. (Dinoflagellata, Syndinea), an intracellular parasite of the ciliate Schmidingerella arcuata: morphology, molecular phylogeny, life cycle, prevalence, and infection intensity. J Eukaryot Microbiol 63:3–15CrossRefGoogle Scholar
- Jung J-H, Kim S, Ryu S, Kim M-S, Baek Y-S, Kim S-J, Choi J-K, Park J-K, Min G-S (2012) Development of single-nucleotide polymorphism (SNP)-based phylum-specific PCR amplification technique: Application to the community analysis using ciliates as a reference organism. Mol Cells 34:383–391CrossRefGoogle Scholar
- Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Mentjies P, Drummond A (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649CrossRefGoogle Scholar
- Montagnes DJS, Lynn DH (1987) A quantitative protargol stain (QPS) for ciliates: method description and test of its quantitative nature. Mar Microb Food Webs 2:83–93Google Scholar